Triploid Atlantic salmon (Salmo salar) may have increased risk of primary field outbreaks of infectious salmon anaemia

Abstract The impact that escaped farmed fish may have on wild populations is of major concern for Atlantic salmon (Salmo salar) farming. Triploid fish, being infertile, were originally introduced to mitigate the genetic impact of escaped fish. In the recent years, an increase in the number of infectious salmon anaemia (ISA) outbreaks in Norway has been observed, mainly in the northern parts, which is also where farming of triploid fish has been licensed. The present study investigated the susceptibility of triploid Atlantic salmon to ISA both by field observations and experimental infections. Based on field observations, we found an increased susceptibility, with 9.4 increased odds to primary ISA outbreaks in triploid fish versus diploid fish at production‐site level, and a tendency of increased odds (3.4) of ISA in triploid fish at individual cage level at sited with primary outbreaks. At some sites, ISA outbreaks were only diagnosed in cages with triploid fish and not in cages with diploid fish. Primary ISA outbreaks are the source for further spread of the disease, and it is noteworthy that in an experimental trial we found significantly more viral RNA in non‐ISA‐vaccinated triploid than in non‐ISA‐vaccinated diploid fish at the peak of the infection. Interestingly, the notable differences of susceptibility to ISA for non‐ISA vaccinated diploid and triploid fish observed in field were not repeated experimentally. The possible increased risk of ISA should be considered when evaluating the costs and benefits of triploid salmon in farming. It is recommended to keep triploid and diploid fish in biosecure separated sites, or that triploid fish are not farmed at all.

Atlantic salmon farming. Triploidization is obtained by high-pressure treatment of newly fertilized salmon eggs. Triploid fish will, due to the extra set of chromosomes, have significantly larger cells than diploid fish. For many species, polyploidy increases body size, but fish compensate by reducing the number of cells and the individual body size appears normal and similar to that of diploid fish. However, triploid salmon differ from natural diploids by requiring more dietary phosphorus during early development, they may have a different gill microbiota, and they have a higher prevalence of eye cataracts (Brown et al., 2021;Olsvik et al., 2020;Sambraus et al., 2020).
Studies of the relative susceptibility to infectious diseases for triploid versus diploid salmon have been limited; however, no differences were found for the susceptibility to infectious pancreatic necrosis virus (IPNV), salmonid alphavirus (SAV) or to severe Neoparamoeba perurans infestation (Chalmers et al., 2017;Moore et al., 2017). Also, vaccination of triploid salmon against furunculosis was found to give good protection (Chalmers et al., 2016). Winter ulcers have been reported to be prevalent in triploid fish that were moved to sea water during the fall (Stien et al., 2019).
The number of annual outbreaks of infectious salmon anaemia (ISA) in Norway has been low to moderate since a large ISA epidemic in the early 1990s. ISA can reduce the economical sustainability for an affected company, the virulent virus may further infect neighbouring farms, it is a notifiable fish disease and outbreaks may affect trade in salmon on the international market. A relatively sharp increase in the annual number of ISA outbreaks, i.e., 23 outbreaks, was recorded in Norway in 2020 and 25 outbreaks were recorded in 2021. The increase in outbreaks has mainly occurred in the northern parts of Norway (Dean et al., 2022), which is the area where farming of triploid fish has been licensed. In an earlier study, an increased risk of primary ISA outbreaks was found when the production site was located at high latitude, i.e., in northern parts (Lyngstad et al., 2018).
The proportion of salmon produced in the two northernmost counties has increased from 14% to about 22% of the total production of Norway over the last 20 years. Studies of infection, disease, and epidemiology of ISA in triploid fish are therefore needed.
The two phenotypic variants of ISA virus (ISAV), the ISAV-HPR0 and ISAV-HPRΔ, non-virulent and virulent, respectively, are named after the highly polymorphic region (HPR) in the hemagglutininesterase (HE) gene. The non-virulent and virulent phenotypic ISAV variants are however, differentiated not only by sequence differences in the HPR of the HE gene, but also by differences in the fusion (F) gene.
The non-virulent HPR0 strain has a strong tropism for gills and is highly prevalent in the sea phase of farmed salmon (Lyngstad et al., 2012). The origin of primary outbreaks of ISA is thought to be a transition from a non-virulent HPR0 to a virulent HPRΔ variant.
Outbreaks assumed to be associated with the transition from nonvirulent HPR0 to ISAV-HPRΔ are often termed primary outbreaks (Christiansen et al., 2017;Nylund et al., 2019). Around 40% of ISA outbreaks in Norway have been considered to be primary outbreaks (Aldrin et al., 2021). If virulent ISAV-HPRΔ from primary outbreaks spreads horizontally through live-animal movements, equipment, or through passive transport via water currents, to nearby farms and causes disease outbreaks, then these outbreaks are termed secondary outbreaks (Lyngstad et al., 2018). In ISA epidemics, secondary outbreaks obviously predominate. Under field conditions, it is often not possible to trace the disease to a known source of infection, and the lack of definite tracking may be the basis for the classification as primary outbreak. Thus, the term primary outbreak in the field also often encompasses outbreaks in which the infection source cannot be identified. In recent years, many sporadic outbreaks of ISA in Norway have been classified as primary outbreaks (Dean et al., 2022).
In the present work, the epidemiology of ISA outbreaks in triploid fish in farms was studied. The results indicate that primary ISA outbreaks have a significantly higher probability of occurring in triploid fish than in diploids. To test the results found in the field study, an experimental susceptibility and vaccination trial was subsequently performed.

| Field outbreaks of ISA
A commercial farming company where production of triploid fish is a large part of total production, had 12 confirmed and two suspected (detection of ISAV-HPRΔ by RT-PCR but negative in all subsequent confirmative tests) ISA outbreaks in the period 2015-2020. These outbreaks were investigated in detail and made the basis for analysis of the specific risk factors for outbreaks of ISA in triploid fish.
All production took place in northern Norway in production areas 10-12. An overview of the outbreaks can be found in Table S1 in Supplementary material.

| Fish stock
During the period 2015-2019, the company had 57 sea transfers into a total of 428 net cages. Diploid fish made up 71.3% and triploid 28.7% of stocked cages. The diploid and triploid fish were kept in the same farms but in separate cages. The fish originated from four different genetic breeds with diploid fish coming from all four breeds and triploid coming from two of those breeds. The fish were not vaccinated against ISA.

| Diagnostics and management of ISA cases
The fish sites of the company had monthly routine control by fish health professionals. Increased mortality led automatically to increased investigations of all cages. Suspicion of ISA due to clinical findings was investigated further by RT-PCR analyses. Heart samples were submitted to PatoGen, Ålesund, Norway, a laboratory accredited for PCR analysis. ISA is a notifiable disease and suspicion of disease was reported to the Norwegian Food Safety Authority, which investigated a suspicion of ISA by their own sampling and analysis with RT-PCR-and histopathologic examination. The regulatory criteria of the handling of the outbreaks were set by the Norwegian Food Safety Authority. In addition, the farming company monitored verified outbreaks by sampling for RT-PCR of individual cages to manage the harvest order of the cages in a best possible way.

| Tracing and origin of ISA-HPRΔ
Sequencing and phylogenetic analysis of ISAV-HPRΔ had been performed by PatoGen, the diagnostic company that served the sites, as a part of their portfolio of analyses. Alignment of the nucleotide sequence of the gene segment encoding the HE gene was the basis of the phylogenetic analysis.

| Experimental fish
The study included one experiment with initial vaccination and subsequent challenge, and one challenge experiment. Both were conducted at VESO Vikan aquatic research facility (Vikan, Norway, internal reference V4596). Diploid and triploid fish used in the study were both of SalmoBreed origin, confirmed free of known salmon pathogens and there was no difference of clinical presentation between triploid and diploid groups before initiation. The design of the study is shown in Figure 1.
Fish were fed according to standard procedures and kept at 12 C temperature during the experiment. The fish n = 527, size 40-60 g, were in fresh water at vaccination and were put on sea water one week before infection, i.e., at week 8 post vaccination (wpv). The experiment was performed with photoperiod 12:12, except for 2-8 wpv when the fish were exposed to 24:00 photoperiod to induce smoltification. Prior to handling, the fish were anaesthetised by bath immersion in benzocaine chloride (1-2 min, 60 mg/L). A concentration of benzocaine chloride of 120 mg/L was used to kill.
The fish were observed minimum once per day. The criteria used to assess humane endpoint were loss of ability to maintain their position in the water, darkening of skin, bleedings and exophthalmia and no evasion to netting. Fish that reached the terminal stage were killed. The water in all experimental tanks were changed from fresh water to sea water one week before ISAV-shedder fish were added. A pre-infection test with three different doses of ISAV, strain AL V321 injected i.p., was performed with diploid fish, 10 fish per group, to estimate susceptibility (Kibenge et al., 2009). Mortality of injected fish began on day 12 at the highest dose, and based on the mortality curves, it was chosen to use the highest dose in the experiment itself.

| Vaccines
The fish were immunized with either a vaccine with specific ISA com- Samples were taken weekly for five weeks from six fish per group until the termination of the experiment.

| Samples
Before vaccination, which took place on Day 0, samples of heart, spleen, kidney and blood from six fish were collected and fixed in RNAlater. Similar samples were taken at the time points described for the individual tanks. Whole blood samples were collected, and the cell pellet and plasma were separated by centrifugation. Weight and length of the fish were measured at all samplings.

| RNA isolation and RT-qPCR
To estimate the level of viral RNA and relative expression of immune response genes, RT-qPCR was run using samples from six fish from each group from each sampling. Total RNA was extracted from 30-40 mg heart and kidney tissue using the RNeasy Mini kit and QIAcube System (Qiagen) as described earlier (Aksnes et al., 2021). The concentration of RNA was determined by spectrophotometry using the Nanodrop ND1000 (Nanodrop Technologies, Wilmington, USA). For each sample, 750 ng of total RNA from kidney samples and 375 ng from heart were subjected to cDNA synthesis using Quantitech® Reverse Transcription (Qiagen); in a total volume of 20 ul. The TaqMan assay (PE Applied Biosystems) was used for qPCR with an input of 5 μl diluted cDNA (equivalent to 15 ng RNA used in cDNA synthesis) per reaction in a total reaction volume of 13 μl. Primers and probe targeting ISAV genomic segment 8 were used as described earlier (Olsen et al., 2012). The qPCR reaction conditions were 300 nM primer, 200 nM probe, 6.5 μl TaqMan®.  Table 1.
The elongation factor (EF1αb) was used as the reference gene (Lovoll et al., 2011). The cycling parameters were 50°C/2 min and 95°C/10 min, followed by 40 cycles of 95°C/15 s, 58°C/15 s and 60°C/1 min. The Cq for EF1αb in kidney was stable in the range 15.2-15.3, and in heart at 16.2-16.3, and the expression of ISAV RNA is therefore, for simplicity, given as a Cq value.

| Statistical analysis
For the experimental data, one-way ANOVA was performed using JMP Pro 15 for windows (JMP Software, Marlow, United Kingdom).
The significant level for rejection of null hypothesis (H 0 ) was set at probability value (p) < .05. The differences of the RT-qPCR results were analysed statistically using Wilcoxon matched pairs signed rank test due to the small sample size (n = 6). For field data odds ratio for primary ISA outbreak at a site were analysed in Stata using exact logistic regression test for triploid fish versus diploid fish (Stata For the challenge study in tank 1, mortality was used as endpoint. Differences between groups were assessed using a Kaplan-Meier survival analysis followed by a Mantel Cox log-rank test. To prove vaccine protection, the AJm7 vaccinated groups had to show significant reduced mortality compared to their respective control group, i.e., AJm6 vaccinated groups. If significant protection of vaccination was observed, similar analyses were performed between triploid and diploid fish groups to assess whether these fish populations showed different protection.

| Field studies
In the period 2015-2020 there were 12 confirmed ISA outbreaks within the company's sea sites. In addition, there were two suspected cases where ISAV-HPRΔ was detected once but ISA was not confirmed in the consecutive testings. ISA occurred in a total of 51 cages in these outbreaks, when including the two suspected cases (one cage each). The proportion of cages with ISA and triploid fish was 25 out of 123 cages (20.3%), while the corresponding proportion for diploid fish and ISA was 26 out of 305 (8.5%). Nine of the 12 confirmed ISA outbreaks were diagnosed more than 12 months after the fish had been put to a sea site. Basic data per year are given in

| Primary ISA outbreaks are more prevalent in triploid fish
Based on phylogenic analysis and tracing of infection, the probable origin of the 12 confirmed ISA outbreaks and the two suspected were categorized as primary or secondary at site level. One outbreak in diploid fish was classified as primary, while six outbreaks were assessed as primary in triploid fish ( Table 2). The odds ratio for primary ISA outbreak at site level in triploid fish versus diploid were found to be 9.4 (p = .047) with confidence interval 1.02-464.

| Triploid fish are more susceptible to ISA
There were two cases where the difference between triploid and diploid fish regarding susceptibility to ISA was clearly demonstrated.
One example was a primary outbreak in 2020 in a fjord system with three separate production sites. These three sites held a total of 15 cages of which four had triploid fish, and all these four cages were in one of the three production sites. In the second example fish were transferred to a sea site of 13 cages where five cages held triploid and eight held diploid fish. ISA was confirmed between 9 and 12 months after sea transfer in all five cages with triploid fish, but not in any of the eight cages containing diploid fish (Figure 3). The triploid smolts originated from three smolt farms, the diploid smolts came from three smolt farms of which one smolt farm was common for both diploid and triploid fish.

| Equal susceptibility to ISAV for non-ISAvaccinated diploid and triploid fish experimentally
Tank 4 was designed to estimate differences in susceptibility to ISAV between non-ISA vaccinated diploid and triploid fish. There was a tendency that the virus was detected in diploids before triploids, with one more ISAV positive sample out of six samples at 10, 11, and 12 wpv in diploids ( Figure 4).
There was no statistically significant difference in viral load, measured as viral RNA, between diploid and triploid groups at any of the sampling times i.e., at week 12 the Cq of groups Diploid AJm6 and Triploid AJm6 were 24.5 ± 5.0 and 26.0 ± 6.6; and at week 13 the Cq were 19.0 ± 0.7 and 18.4 ± 3.8, respectively (Figure 5a). This differs from Tank 2 and 3 where there was significantly (p < .05) more virus in Triploid AJm6 than in Diploid AJm6.

| Innate immune responses in diploid and triploid fish to ISAV are similar
The innate antiviral response to ISAV in non-ISA-vaccinated diploid and triploid fish was assessed by measuring the relative expression After infection with ISAV the expression of Mx, Vip and IFN-1 genes increased following the load of viral RNA, with only minor difference in expression levels between diploid and triploid groups ( Figure 5b).

| Vaccination protects against mortality
The mortality of the virus shedders started ten days after being added to the Tank 1 and they were all dead 15 days after addition.
Fish in the vaccinated groups started to die on day 22-23 after the addition of the virus shedders.
In the groups Diploid AJm6 and Triploid AJm6, i.e., the groups that received the vaccine without a specific ISA component, the mortality was 100% (Figure 6).
In the groups Diploid AJm7 and Triploid AJm7, i.e., the groups that received the vaccine with a specific ISA component, the cumulative mortality was 57% and 82%, respectively. For both groups, mortality was statistically significantly lower (p < .0001) than for the groups Diploid AJm6 and Triploid AJm6 ( Figure 6). That is, the vaccine had a statistically significant protective effect against mortality for both diploid and triploid groups.
We found that the difference in mortality between the groups Diploid AJm7 and Triploid AJm7 was also statistically significantly different (p < .05) ( Figure 6) with a better effect in the diploid group.
This may indicate a difference in the effect of vaccination, and/or a more severe ISAV infection in the triploid fish.

| Are viral RNA loads higher in non-ISAvaccinated triploid fish?
At the peak of the infection at 13 weeks post vaccination (wpv), i.e., four weeks after addition of shedder fish, there was significantly F I G U R E 3 A specific field outbreak of ISA. ISA was confirmed between 9 and 12 months after sea transfer. All five cages with triploid fish became ISA positive, while the eight cages with diploid fish did not. Blue = diploid fish. Green = triploid fish. White = empty cage. Red perimeter = ISA confirmed. Blue perimeter = ISA not confirmed. more viral RNA (p < .05) in Triploid AJm6 than in Diploid AJm6 in both heart and kidney in Tanks 2 and 3 (data not shown). This indicates that more viral RNA is produced in non-ISA-vaccinated triploid than in non-ISA-vaccinated diploid fish. The amount of viral RNA was very high with Cq ≈ 13-14 in heart at this sampling time, as was reflected by that all the fish in the Ajm6 groups had died by week 15 ( Figure 7). As mentioned above, there was no statistically significant difference in viral load, measured as viral RNA, between Triploid AJm6 than in Diploid AJm6 groups in Tank  3.4.6 | Viral RNA loads are higher in heart than in kidney for all groups At the peak of the infection at 13 wpv there was statistically significant (p < .00001) more virus in the heart than in the kidney for all groups in Tanks 2, 3 and 4 (Figure 7).

| DISCUSS ION
The possible negative impact that escaping fish may have on wild fauna is a major concern of Atlantic salmon farming. Triploid fish, being infertile, has been introduced to reduce the genetic impact escapees may have on wild populations of Atlantic salmon. In the last years there has been an increase in the number of ISA outbreaks, mainly in the northern parts of Norway that also is the area where farming of triploid fish has been licensed. The present study investigated the suspected increased susceptibility of triploid Atlantic salmon to ISA.
In the field part of this study, we found an increased risk for primary outbreaks of ISA with a 9.8 odds ratio for primary outbreaks in triploid fish versus diploid fish at site level, and an esti- F I G U R E 6 Accumulated mortality in Tank 1 after addition of shedder fish. Statistical analyses were performed by plotting the survival curve for the individual experimental groups (Kaplan-Meier survival analysis) and potentially significant differences between groups were tested by a log-rank (Mantel-Cox) test (software: GraphPad Prism v7.03). Only significant analyses are depicted. N = 30 fish per group. *p < .05, ****p < .0001.

F I G U R E 7
Cq values for ISAV in heart and kidney tissue of diploid and triploid fish, (a) vaccinated without ISAV component (AJm6)  The fish were smoltified, and stress was experimentally reduced by replacing fresh water with sea water a week before infection. This simulates a natural situation, but since the fish were exposed to virus short after smoltification, it was considered as a relatively tough ex- Triploid fish have three sets of chromosomes and 1.5 times more genetic material than diploid fish. Cells in triploid fish are therefore larger and have a lower surface area to volume ratio, which may impair gas exchange (Riseth et al., 2020). Fish erythrocytes are nucleated, and it can be speculated whether the larger blood cells in triploid fish result in poorer blood flow in peripheral capillaries and contribute to poorer wound healing. Earlier it has been found that triploid Chinook salmon (Oncorhynchus tshawytscha) have reduced oxygen-carrying capacity of the blood (Bernier et al., 2004). Experimental findings have shown that triploid salmon are more sensitive to low oxygen saturation at high water temperatures (Hansen et al., 2015), and that triploid salmon have reduced gill surface (Sadler et al., 2001).
In a previous study, it was found that triploid salmon in northern parts of Norway released into the sea in November-December had a higher incidence of skin ulcers (Stien et al., 2019), which is in line with observations of fish health professionals with long experience with triploid fish that observe more winter wounds in triploid than diploid fish (L. Martinsen, personal communications) It has also been noted that triploid fish have a higher prevalence of melanin spots in muscles (Larsen et al., 2014). The results of our study support the assumption that triploid fish are generally more susceptible to disease than diploid fish.
In our data 9 of the 12 confirmed ISA outbreaks occurred more than 12 months after transfer to sea, which indicated that long production time at sea increases the risk to develop ISA. This is in line with earlier findings where long production period in sea was associated with increased risk of primary ISA outbreak (Lyngstad et al., 2018). This was explained with long production period causing increased probability of exposure to both ISAV-HPR0 and ISAV-HPRΔ.
At the peak of the infection in the experimental challenge, we found more viral RNA in the heart than the kidney in both diploid and triploid fish. The virus replicates primarily in endothelial cells (Aamelfot et al., 2014), and the findings indicate that samples from heart contain more remnants from endothelial cells than samples from kidney do.

| CON CLUS IONS
We found that triploid Atlantic salmon may have increased risk of primary field outbreaks of ISA. But we found no indications for a difference in susceptibility to an ISAV virulent strain for non-ISA vaccinated diploid and triploid fish in an experimental setting.
There was better survival, and lower viral RNA load, in ISA-

ACK N OWLED G EM ENTS
Øystein Wessel read and commented manuscript thoroughly and helped with the figures. The research was funded by Norway Royal Salmon (NRS) and Pharmaq.

CO N FLI C T O F I NTE R E S T
LM is an employee of NRS, and MMS-K and ASL are employees of Pharmaq. They declare no conflict of interest. The other authors declare no conflict of interest. The funders had no roles in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.